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Method for the generation of monoclonal antibodies derived from human b cells

a monoclonal antibody and human b cell technology, applied in the field of human b cells, can solve the problems of low cloning efficiency, elusive efficient method of obtaining such antibodies, and low efficiency of immortalization

Inactive Publication Date: 2013-01-31
DUKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a way to make and clone human B cells, which are important for making antibodies. The invention also includes methods to use these monoclonal antibodies for treating and diagnosing diseases.

Problems solved by technology

An efficient method of obtaining such antibodies has, however, been elusive.
The efficiency of immortalization, however, can be low, as can the cloning efficiency of the immortalized B cells.

Method used

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  • Method for the generation of monoclonal antibodies derived from human b cells
  • Method for the generation of monoclonal antibodies derived from human b cells
  • Method for the generation of monoclonal antibodies derived from human b cells

Examples

Experimental program
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example

Experimental Details

Preparation of Complete Medium

[0029]Complete Medium (CM) for PBMC, EBV-B cells, J774A.1, and K6H6 / B5 cell lines

[0030]RPMI 1640 (Invitrogen) supplemented with:[0031]1) 15.2% heat-inactivated fetal calf serum (FCS)[0032]2) 1% non-essential amino acids (NEAA)[0033]3) 1 mM sodium pyruvate[0034]4) 15 mM HEPES buffer, pH 7.3[0035]5) 2 mM L-glutamine (Glu)[0036]6) 100 U / ml penicillin G (Pen)[0037]7) 100 μg / ml streptomycin (Strep)

[0038]Preparation:[0039]1. Thaw a bottle of FCS (500 ml / bottle, −20° C.) and incubate at 56° C. for 30 min for heat inactivation (inactivation of c′)[0040]2. Let bottle cool down and add 31 ml of Glu+Pen / Strep (100×, 100 ml / bottle, −20° C.)[0041]3. Aliquot the FCS-antibiotics mixture into tubes at 50 ml / tube and freeze at −20° C.[0042]4. Add 100 ml of FCS-antibiotics mixture to bottle of RPMI 1640 (500 ml / bottle, 4° C.)[0043]5. Add 6.2 ml of HEPES buffer (100×, 100 ml / bottle, 4° C.) into the bottle[0044]6. Add 6.2 ml of sodium pyruvate (100×, 1...

experiment 8-28

[0136]

FrequencySampleof positiveCDR3IDPCR wellsVDHJMutatedlengthProductive113 / 20 1~69*023~3*014*020.0372499F211 / 20 4~39*066~13*011*010.04301015F1 / 203~49*032~2*01 / inv, 02 / inv4*020.0925372N34 / 203~23*012~2*01 / inv, 02 / inv3*020.02949117F43 / 204~39*012~21*025*010.05221919F53 / 202~5*104~17*014*020.04143613F613 / 20 4~31*030~IR*01C4*020.02193810F73 / 204~34*012~2*01, 024*020.00266718F88 / 204~59*012~2*024*010.03753418FTubeOriginalSortCDR3IgIDWellWellVDHJMutatedlengthIsotypeProductive1B7A101~69*023~3*014*020.0374639779G1F1B7A101~69*023~3*014*020.0372492849G1F1B7A101~69*023~3*014*020.0373563229G1F1B7A21~69*023~3*014*020.0401146139G1F1B7A21~69*023~3*014*020.0401146139G1F1B7A21~69*023~3*014*020.0402298859G1F1B7A31~69*022~2*01, 021*010.0744985679MN1B7A31~69*023~3*014*020.0372492849G1F1B7A31~69*023~10*01 / inv1*010.0442477889A1N1B7A41~69*023~3*014*020.0372492849G1F1B7A41~69*023~3*014*020.0372492849G1F1B7A41~69*023~3*014*020.0373563229G1F1B7A51~69*023~3*014*020.0429799439G1F1B7A51~69*023~3*014*020.037249284...

experiment 8-29

Experiment 8-30—Total IgG ELISA

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Abstract

The present invention relates, in general, to human B cells, and, in particular to a method of immortalizing and cloning human B cells and to monoclonal antibodies derived therefrom. The invention further relates to methods of using the monoclonal antibodies for therapeutic and diagnostic purposes.

Description

[0001]This application claims priority from U.S. Provisional Application No. 61 / 322,725, filed Apr. 9, 2010 and U.S. Provisional Application No. 61 / 322,821 filed Apr. 10, 2010, the entire contents of which are incorporated herein by reference.[0002]This invention was made with government support under Grant No. AI067854-02 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD[0003]The present invention relates, in general, to human B cells, and, in particular to a method of immortalizing and cloning human B cells and to monoclonal antibodies derived therefrom. The invention further relates to methods of using the monoclonal antibodies for therapeutic and diagnostic purposes.BACKGROUND[0004]The isolation and characterization of monoclonal antibodies that neutralize, for example, a broad spectrum of HIV-1 isolates are important to the design of an effective HIV-1 vaccine. An efficient method of obtaining such antibodies has, h...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86
CPCC07K16/00C07K2317/76C07K16/1045
Inventor HAYNES, BARTON F.BONSIGNORI, MATTIAHWANG, KWAN-KILIAO, HUA-XINMONTEFIORI, DAVID C.LUFTIG, MICAH A.
Owner DUKE UNIV
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